The primary objective of this proposed work is the development of fundamental links between the nature of the protein folding free energy landscape and the underlying mechanism and kinetics of protein folding. Key questions to be addressed during this funding period include: (i) What is the relationship between the topology folded protein and the free energy landscape, which determines the general mechanism of folding? (ii) How does the balance between local and longer range (in sequence) interactions change to provide stabilization of the protein structure throughout the folding process? (iii) How is it altered with changing temperature? (iv) How do changes in the balance of long range and short range interactions yield differing mechanisms of folding, or in extreme cases lead to misfolding and problems attendant with such conformational distributions? (v) What is the nature of fluctuations between different conformations within the same region of configuration space; e.g., what are the barriers associated with interconversion between different conformations within a given region of a folding reaction coordinate? (vi) What role does water play in mediating protein-protein interactions throughout the folding process? Using molecular dynamics simulations with specialized sampling techniques in explicit and implicit solvent representations, we will explore the folding process for a number of proteins. Langevin dynamics methods will be utilized to explore kinetics and thermodynamics of minimalist off-lattice models for proteins of differing topology. Theoretical models will be developed to express the general features of thermodynamics and kinetics on protein folding landscapes. Our focus will be on small single domain proteins of differing topology, but we will also extend aspects of this work to more complex multi-domain proteins and proteins with intermediates. We will (i) augment our analysis of the folding of helical proteins by examining the engrailed homeodomain and (ii) extend our studies of alpha/beta proteins by studying the folding landscape versus temperature for segment B 1 of streptococcal protein G and peptostreptococcal protein L at both atomic and 'minimalist" detail. We will (iii) extend our analysis of all beta proteins to the Cold Shock protein family, CspA and CspB, the src-SH3 domain and the WW domain. Additionally, (iv) we will explore the direct folding of small engineered polypeptides at atomic detail using implicit solvent, with the generalized Born model, and advanced sampling methods such as replica-exchange and related variants. Comparisons between key interactions anticipated during folding (based upon our calculations) and observations from folding studies on these and similar proteins will be made. (v) Simplified (off-lattice minimalist) models will be developed and used to examine general features of different folding scenarios. We will explore the balance of non-specific versus specific interactions and the role of topology in folding thermodynamics and kinetics for such models. These calculations will complement the all atom calculations by providing both methodological insights and ideas to fuel analysis and calculations on detailed models.